In a trace substance assay of hormone, tumor marker, infectious pathogen marker, infective pathogen antibody, etc. for blood, serum, plasma, or body flood as a sample or a specimen, there has generally been used a method of qualitatively or quantitatively detecting proteins, etc. in the sample or the specimen by bonding based on antigen-antibody bonding reaction on every analysis items of proteins. The antibody or the antigen bonding to a protein as an object of analysis in this case is bonded with a radioactive isotope, fluorescent dye, luminescent dye, enzyme, rare earth complex, metal ion, etc. and referred to as a labeled antigen, a labeled antibody, or a tracer.
Similarly, for the assay of DNA or RNA, for example, of a pathogen, a drug metabolizing marker, etc. in the sample or the specimen, a DNA chain or an RNA chain complementary to the DNA or RNA of an object of analysis is hybridized to detect the object of analysis. In this instance, the complementary DNA chain or RNA chain is labeled directly or indirectly with a fluorescent substance or a luminescent substance.
Immunoassay is a specific measuring method of a biological substance on the basis that an antigen and an antibody bonded thereto form antigen-antibody bonding. The method includes a solution precipitation reaction method, a carrier agglutination reaction method, and a labeled antibody method based on the principle of measurement. The solution precipitation reaction method is a method of optically measuring and determining agglutinates formed by the antigen-antibody bonding in a solution, for which immuno turbidimetry, immuno nephelometry, etc. are known. The carrier agglutination reaction method is a method of subjecting a carrier such as an antibody-immobilized latex and a sample solution (antigen) to antigen-antibody bonding and measuring and determining the resultant agglutinates of the carrier optically or imagewis. The carrier agglutination reaction method measures apparent particle diameter or decrease or increase in the transmission light by latex turbidimetry, a latex nephelometry, particle counting, image processing, etc. The labeled antibody method is a method of using an antibody labeled with various labeling substances to subject the same to antigen-antibody bonding, and measuring only the ingredient reacted with the labeled antibody. The labeled antibody methods known include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, chemiluminescent immunoassay, electroluminescent immunoassay, etc. based on the labeling substances.
Problems pointed out for the immunoassay are to be set forth below.
1. Effect by heterophil antibody: In the system where a monochronal antibody is used as the antibody, when HAMA (human anti-mouse antibody) is present in the sample, it crosslinks a labeled antibody and an immobilized antibody, resulting in false-positive.
2. Non-specific reaction with blocking agent, etc.: A blocking agent or a protective protein contained in a reagent reacts with substances in the sample, resulting in false-positive.
3. Non-specific reaction with labeled enzyme: When a substance bonding to a labeled enzyme is present in the sample, background increases.
4. Prozone phenomenon or high dose huck: When the concentration of an antigen is higher than a measurement range or when the concentration of a labeled antibody greatly exceeds the measurement range, false-negative occurs. In an antigen excessive region, an antigen-antibody complex becomes soluble to sometimes show an abnormally low value.5. Effect of hemolysis: When hemolysis in the sample is intense, this results in influence on the color formation upon judgment to make the judgment difficult.6. When faces and urine are used as the sample: Fresh samples are used. Samples containing intense turbid urea or blood cells are not suitable.7. Effect due to the property of sample: When the viscosity of the sample, particularly, the concentration of protein (M protein, cryoglobulin positive sample) is high, the reaction rate is low and false-negative occurs. Test is performed again by using a diluted sample. Non-specific scattering sometimes occurs due to the presence of milky serum or immuno complex.8. False-positive and false-negative due to crossed reaction: They are observed in a system in which a recombinant antigen is used. Further, false-positive is shown by crossed reaction in FSH, TSH, and LH.9. Effect of drugs used (atropine, caffeine, acetoamidephenol, acetylsalicylic acid, ascorbic acid, etc.): False-positive is shown due to the effect of drugs in a urine hCG measuring system.10. Inter-reagent difference of measuring sensitivity: In some cases, while the reaction shows negative for a predetermined judging time, positive reaction develops with lapse of time.
Long time has been elapsed since Berson and Yalow completed radio immunoassay by labeling radioactive isotopes to antigens or antibodies. This is used still at present as a useful method in view of the good sensitivity and high specificity. Meanwhile, development and improvement have been made for enzyme immunoassay, fluorescent immunoassay, luminescent immunoassay, etc.
The enzyme immunoassay is a method of labeling an enzyme, instead of the radioactive isotope, to an antigen or an antibody, which enables measurement at high sensitivity in view of the high catalytic activity of the enzyme to a substrate.
A time-resolved fluorometry of pulsatively irradiating a final product of antigen-antibody reaction with exciting light and, after lapse of a time in which fluorescence generated from a reaction vessel, etc. is quenched, measuring the fluorescence of the substance has been developed. In recent years, europium complexes or samarium complexes having relatively long fluorescence quenching time or not requiring sensitizer have been developed and the method has become more effective.
In the luminescent immunoassay, a method of labeling isoluminol or acridinium ester and measuring the amount of luminescence is provided, and measuring systems according to following combinations are established: a combination of peroxidase and luminol using a substrate capable of luminescent reaction while using an enzyme as the labeling substance, or a combination of a substrate such as AMPPD that emits light when a phosphoric acid group is dissociated and an alkali phosphatase, or a combination of using luciferase as a labeling substance and using luciferin as a substrate.
In labeled antigen, labeled antibody, labeled DNA, labeled RNA, tracer, etc., while a single fluorescent substance or luminescent substance is bonded to a material to be labeled, it has been attempted to bond many fluorescent substances or luminescent substances to a material to be labeled for the purpose of increasing the amount of fluorescence or luminescence. A plurality of fluorescent substances or luminescent substances are formed as a complex, as a complex with bovine serum albumin, streptavidin or like other protein, or bonded to the surface of a fine particle, and they are bonded as a labeling material to the material to be labeled. For the fine particle, particles of gelatin, latex, or polystyrene having a diameter of about 2 micrometer (μm) are generally used, serving as a carrier for the fluorescent substance or the luminescent substance. In recent years, finer particles of about 100 nanometer (nm) diameter have also been developed. Further, for the bonding or immobilization of the fluorescent substance or the luminescent substance to the carrier, the fine particles and the fluorescent substance or the luminescent substance are often mixed in a buffer solution and bonded by electric bonding.
The reaction surface as a site for the antigen-antibody reaction or gene detection method generally comprises a flat plate or particle of a plastic or glass material, and magnetic particle incorporating iron oxide is also used. Further, filter, woven fiber, multi-layered paper, porous film, etc. are sometimes used for increasing the surface area for immobilization.
When individual bodies having the reaction surface are particles, they are put into a reaction vessel and mixed with a sample or a specimen or a reagent, and cells, microtiter plates, etc. made of transparent mineral ores such as quartz or artificial stone thereof, glass, or compounds such as polycarbonate material, polystyrene material, and polyvinyl material are used as the vessel.
A method of measuring items in immuno serum assay for clinical inspection often includes a method of detecting an antigen-antibody reaction by using an antibody or an antigen having a fluorescent substance or a luminescent substance as a labeled substance by utilizing the reaction. In this case, the antigen-antibody reaction is detected also even if the fluorescent substance or the luminescent substance such as a dye, fluorescent dye, or phosphorescent dye is labeled directly to the antibody or the antigen. However, when labeled antibody or antigen is prepared by bonding a plurality of substances to a labeling carrier thereby forming a complex of a fluorescent substance or a luminescent substance and a carrier and binding the same to an antigen or antibody, the number of the fluorescent substance or the labeled body bonding to one molecule of the antigen or the antibody can be increased to generate more intense signals. When proteins such as bovine serum albumin and keyhole limpet hemocyanine, particles of plastics, for example, of polystyrene and polypropylene, ferrite, silica, gelatin and other polymerization products, or chained carbon compounds such as polyvinyl alcohol are used, they correspond to the labeling carrier.
Alternatively, a plurality of fluorescent substances or luminescent substances can be sealed in liposomes, lipid films, or hollow plastic materials thereby capable of amplifying signals. In this case, the liposomes, lipid films, or hollow plastic materials correspond to the carrier.
The carrier is formed as a complex with the fluorescent substance or the luminescent substance and used as a labeling material. The labeling material sometimes forms a macro agglutinate block during preservation. Further, the thermal stability lowers remarkably by the formation of the complex, and the material also tends to be adsorbed easily to a reaction vessel or the reaction material for immobilization to result in increase of non-specific background. In the same manner, due to the property of the carrier and the labeling substance and, further, the ingredients contained in the solution, extinction or quenching occurs that reduces color formation, luminescence or fluorescence of a substance. Further, when the antigen or the antibody is labeled, since the molecule of the labeled material is large, the antigen-antibody reaction is remarkably lowered.
In the antigen-antibody reaction by fluorometry or luminometry, the fluorescent substance, the luminescent substance, or the enzyme bonded to the labeled antibody or the labeled antigen is not bonded to the antibody or the antigen in 1:1 relation but a plurality of molecules of the fluorescent substance, the luminescent substance, or the enzyme are generally bonded to one molecule of the antibody or the antigen. It is intended to takeout signals under amplification.
The fluorescent substance, the luminescent substance, or the enzyme is bonded in plurality to other protein or support, and the protein or the support is used as the carrier for the fluorescent substance, etc. It is important that the carrier satisfy the following requirements.
1. Preferably, the carrier has a large surface area while the molecular weight is low and has a specific gravity about equal to that of a buffer to be used.
2. Preferably, the carrier tends to immobilize the fluorescent substance, etc. and does not lower the fluorescence or the luminescence of the fluorescent substance, etc.
3. Preferably, the carrier less adsorbs to a reaction vessel or other particles after labeling of the fluorescent substance, etc. to the carrier.
4. Preferably, the carrier is fine and has such a size not to optically interrupt excitation light and, in the same manner, does not hinder the detection of the fluorescence or the luminescence at a photomultiplier or the like. If an object shorter than a wavelength is present, the carrier does not interrupt the light. When the carrier is shorter than the wavelength of the light to be used, the light transmits the carrier without decay when it does not transmits the carrier. Further, light is not scattered by the carrier and generation of noises is minimized.
5. Preferably, the carrier is colorless and transparent and does not hinder the transmission of excitation light. Preferably, the carrier does not generate non-specific fluorescence or luminescence by the irradiation with the excitation light.
6. Preferably, the carrier has high hydrophilicity and high dispersibility. In the same manner, it preferably maintains high hydrophilicity and dispersibility easily after labeling the fluorescent substance, etc.
A homogeneous measuring method (homogeneous assay) is a method as a method in which measurement is always performed in a solution state. As measurement at high sensitivity has been demanded, however, a method of reacting an antigen and an antibody, separating a free labeled antibody (or antigen) not participating in the reaction by a cleaning operation (B/F separation) and then measuring the antigen-antibody complex, that is, a heterogeneous measuring method (heterogeneous assay) has been utilized generally. At present, a homogeneous assay at high sensitivity has become possible by developing a combination of a fluorescent dye and its quencher (quenching dye), or an FRET method of detecting the fluorescence of a second substance due to excitation by energy transfer from a first fluorescent substance to the second fluorescent substance, an LOCI method of applying oxygen channeling, a gold particle method of measuring different color formation due to approaching and agglutination of colloidal gold particles, etc.
Patent document 1 shows a method of disposing first metal particles and second metal particles on a substrate at a distance not causing interaction with each other, supplying the second metal particles having the first metal particles and a first substance bonded to the first metal particles and bonded to a second substance to the substrate, bonding the first metal particles and the second metal particles by way of the bonding between the first substance and the second substance, causing interaction to each other, and observing the bonding between the first substance and the second substance. Patent document 2 shows a method of quantitatively determining the amount of a specimen to be detected by measuring the fluorescence intensity based on the antigen-antibody reaction between a detected specimen-dye complex of a specimen to be detected (antigen) bonded with a not-fluorescent dye and an antibody to the detected specimen-dye complex which recognizes both of the specimen to be detected and the dye as epitopes, and the detected specimen-dye complex and the antibody.
Any of the methods is far from practical use for measuring a specific reaction while realizing high sensitivity and a further development is necessary, since it is necessary to provide special metal particle, substrate, dye, and antibody that recognizes two epitopes, and special techniques are required in the preparation of the system.